Nanoparticles encapsulating soluble biologics, therapeutics, and imaging agents

ABSTRACT

An “inverse” precipitation route to precipitate aqueous soluble species with copolymers as nanoparticles having a hydrophilic, polar core and a less polar shell is described.

This application is a continuation of prior application Ser. No.16/253,850, filed Jan. 22, 2019 (published as U.S. Patent ApplicationPublication No. US 2019-0151252 A1 on May 23, 2019), which is a divisionof prior application Ser. No. 15/321,588, filed Dec. 22, 2016 (publishedas U.S. Patent Application Publication No. US 2017-0209386 A1 on Jul.27, 2017), which is a National Stage of International Application No.PCT/US2015/036060, filed Jun. 16, 2015 (published as InternationalApplication Publication No. WO/2015/200054 on Dec. 30, 2015), whichclaims the benefit of U.S. Provisional Application No. 62/016,363, filedJun. 24, 2014, all of which are hereby incorporated by reference intheir entireties herein.

FIELD OF THE INVENTION

The present invention relates to a process of making nanoparticleshaving a hydrophilic core.

BACKGROUND OF THE INVENTION

Protein and peptide therapeutics are a growing segment of thepharmaceutical marketplace. In eleven years, from 2001 to 2012, theglobal sales of pharmaceutical biologic therapeutics (biologics) morethan tripled from $36 billion to $163 billion. In that same period,revenue generated by biologics within the top 10 selling pharmaceuticalsincreased from 7% to 71% (S. Peters, Biotech Products in Big PharmaClinical Pipelines Have Grown Dramatically, Tufts CSDD Impact Report. 15(2013) 1). Their specificity makes them ideal therapeutics for thetreatment of a variety of diseases including cancer and AIDS. Thisspecificity comes as a result of structural complexity, which is astrength of biologics for use as therapeutics and a challenge in tryingto formulate and deliver them (S. Mitragotri, P. A. Burke, R. Langer,Overcoming the challenges in administering biopharmaceuticals:formulation and delivery strategies, Nat Rev Drug Discov. 13 (2014)655-672).

While humanized antibodies may be long circulating, proteins andpeptides can be cleared from the bloodstream in a matter of minuteseither due to renal clearance or enzymatic degradation (A. K. Sato, M.Viswanathan, R. B. Kent, C. R. Wood, Therapeutic peptides: technologicaladvances driving peptides into development, Curr. Opin. Biotechnol. 17(2006) 638-642). Therefore, delivery and extended release can requireencapsulation of the biologic into nanocarriers (NCs) or microcarriers(MCs). NCs can be defined as having sizes below 400 nm, making themprospects for injectable formulations, and MCs can be defined as havingsizes above 1-10 microns, so that they are appropriate for depotdelivery. Requirements of NCs and MCs are high loading, highencapsulation efficiency, and an appropriate release profile of thebiologic therapeutic.

The term “biologic” can encompass a range of therapeutics includingpeptides, oligonucleotides, polypeptides, polypeptide antibiotics,proteins, and antibodies. For example, a peptide may include a sequenceof 1 to 40 amino acids. While there have been recent promising advancesin oral delivery of biologics, the difficulty in translocating NCsthrough mucus layers and across the GI (gastrointestinal) tractepithelial layer makes this a less developed area than parenteraladministration. However, the principles for NC formulation apply equallyto oral or parenteral NCs. Examples of carriers include hydrogelcarriers composed of water soluble polymers and non-swellable carrierscomposed of hydrophobic or solid matrices.

SUMMARY

A method of the invention for encapsulating water soluble moleculesusing rapid, controlled precipitation is presented. Water solublemolecules—including peptides, proteins, DNA, RNA, non-biologictherapeutics, polysaccharide-based therapeutics (e.g., tobramycin) andimaging agents—precipitate into nanoparticles that are protected by acopolymer stabilizing agent. These particles may be covalently ornon-covalently stabilized. The particles may be coated with anamphiphilic polymer, or processed into microparticles or largermonoliths. Post processing on the final construct may conducted.

A method of the invention for encapsulating a water soluble agentincludes dissolving the water soluble agent and a copolymer in a polarprocess solvent to form a first process solution. The first processsolution can be continuously mixed with a nonprocess solvent to form amixed solution from which a nanoparticle assembles and precipitates. Thecopolymer can include at least one region that is more polar and atleast one region that is less polar. The nonprocess solvent is or mustbe less polar than the polar process solvent. The nanoparticle caninclude a core and a shell. The core can include the more polar regionof the copolymer and the water soluble agent. The shell can include theless polar region of the copolymer. The mixing can cause no more than 20percent by volume of the polar process solvent to phase separate.

In a method of the invention, the water soluble agent can be a biologicmaterial, an amino acid, a peptide, a protein, DNA, RNA, a saccharide,glutathione, tryptophan, a lysozyme, glucagon-like peptide-1 (GLP-1), asmall molecule therapeutic, tobramycin, vancomycin, an imaging agent,eosin Y, tartrazine, a metal chelate, a gadolinium chelate, gadoliniumdiethylene triamine pentaacetic acid (GD-DTPA), or combinations.

For example, the copolymer can be a random copolymer, a block copolymer,a diblock copolymer, a triblock copolymer, a multiblock copolymer, or abranched-comb copolymer. For example, the copolymer can include at leastone more polar region (region that is more polar). The at least one morepolar region of the copolymer can include at least one anionic morepolar region. For example, this anionic more polar region can includeanionic residues (units or monomers), poly(acrylic acid) (PAA),hyaluronic acid, poly(glutamic acid), poly(aspartic acid), orcombinations.

For example, the at least one more polar region of the copolymer caninclude at least one cationic more polar region. For example, thiscationic more polar region may include cationic residues, such aschitosan polymer domains, histadine lipids, histamines, spermadines,polyethylene-imines, or combinations. For example, the copolymer caninclude at least one less polar region (region that is less polar) thatincludes poly(n-butyl acrylate) (PBA), poly(lactic acid) (PLA),poly(caprolactone) (PCL), poly(lactic-co-glycolic acid) (PLGA), lipid orphospholipid grafted units, or cholesterol grafted units, orcombinations. For example, the copolymer can be poly(acrylicacid)-block-poly(n-butyl acrylate) (PAA-b-PBA).

In a method of the invention, the polar process solvent can be water, analcohol, methanol, ethanol, acetone, acetonitrile, dimethyl sulfoxide(DMSO), dimethylformamide (DMF), N-methyl pyrrolidone (NMP), orcombinations.

In a method of the invention, the nonprocess solvent can be chloroform,dichloromethane, an alkane, hexane, an ether, diethyl ether,tetrahydrofuran (THF), toluene, acetone, or combinations. For example,the nonprocess solvent can be chloroform, acetone, or combinations. Forexample, the polar process solvent and the nonprocess solvent can bemiscible.

In a method of the invention, a time of mixing of the process solutionwith the nonprocess solvent is less than an assembly time of thenanoparticle. For example, the water soluble agent and the copolymer canhave a supersaturation level in the solution ranging from 10 to 10,000.For example, the nanoparticle can have a size ranging from about 40 nmto about 400 nm.

A method of the invention includes stabilizing the nanoparticle corethrough crosslinking of the copolymer. For example, the nanoparticle canbe crosslinked during assembly of the nanoparticle. For example, thenanoparticle can be crosslinked after assembly of the nanoparticle. Thecrosslinking can be covalent crosslinking. The crosslinking can benon-covalent, ionic, chelation, acid-base, or hydrogen bondingcrosslinking.

A crosslinking agent can be added to crosslink the copolymer. Forexample, the crosslinking agent can be added to crosslink a portion ofthe copolymer of anionic functionality. For example, the crosslinkingagent can be an alkaline earth halide, a magnesium halide, magnesiumchloride, a calcium halide, calcium chloride, a transition metal halide,an iron halide, iron(III) chloride, spermine, or combinations. Forexample, the crosslinking agent can be a metal acetate, an alkalineearth acetate, a transition metal acetate, calcium acetate, orcombinations. For example, the crosslinking agent can be chromium(III)acetate, or another chromium (III) salt. For example, the water solubleagent can include tobramycin and the tobramycin can crosslink thecopolymer. Other bio-compatible multi-cationic water soluble agents maybe used as crosslinking agents, for example, to crosslink anionicsections of the copolymer.

If the polar agent includes cationic functional groups, thencrosslinking may be achieved by the addition of poly-anionic components.Examples of these are poly(acrylic acid) (PAA), hyaluronic acid,poly(glutamic acid), poly(aspartic acid), citric acid, polycitric acid,anionic oligonucleotides, and multi-valent anions.

A method of the invention includes coating the nanoparticle with anamphiphilic polymer, the amphiphilic polymer including at least onehydrophilic region and at least one hydrophobic region. The amphiphilicpolymer can be dissolved in a water-miscible organic solvent to form asecond process solution. The nanoparticles can be dissolved, suspended,or otherwise included in the second process solution. The second processsolution can be continuously mixed with an aqueous solvent to form asecond mixed solution from which a coated nanoparticle assembles andprecipitates. The coated nanoparticle can include a core, a shell, and acoating. The coating can include an inner region and an outer region.The inner region can include the at least one hydrophobic region of theamphiphilic polymer. The outer region can include the at least onehydrophilic region of the amphiphilic polymer.

The amphiphilic polymer can be a random copolymer, a graft copolymer, ablock copolymer, a diblock copolymer, a triblock copolymer, or amultiblock copolymer. For example, the amphiphilic polymer can bepolystyrene-block-poly(ethylene glycol) (PS-b-PEG), poly(lacticacid)-block-poly(ethylene glycol) (PLA-b-PEG),poly(caprolactone)-block-poly(ethylene glycol) (PCL-b-PEG),poly(lactic-co-glycolic acid)-block-poly(ethylene glycol) (PLGA-b-PEG),poly(ethylene oxide)-block-poly(propylene oxide)-block-poly(ethyleneoxide) (PEO-b-PPO-b-PEO), or poly(ethylene oxide)-block-poly(butyleneoxide)-block-poly(ethylene oxide) (PEO-b-PBO-b-PEO).

The water-miscible organic solvent can include tetrahydrofuran (THF)and/or acetone and the aqueous solvent can be water.

In an embodiment of the invention, a nanoparticle can include a morepolar region of a copolymer and a water soluble agent and a shellincluding a less polar region of the copolymer. The more polar region ofthe copolymer in the core can be crosslinked. The nanoparticle can alsoinclude a coating. The coating can include an inner region and an outerregion. The inner region can include a hydrophobic region of anamphiphilic polymer. The outer region can include a hydrophilic regionof the amphiphilic polymer.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates steps in the production of nanoparticles according tothe invention. An “inverse” nanoparticle can be formed of a copolymerthat assembles into a nanoparticle having a hydrophilic core including awater soluble agent and a less polar shell 201. The core can becrosslinked to form an “inverse” nanoparticle with a crosslinked core301. The “inverse” nanoparticle can be coated with an amphiphilicpolymer to produce a structure that is stable in aqueous solution 401.

FIGS. 2A, 2B, 2C, and 2D illustrate how the variation of parameters inthe Flash NanoPrecipitation process can control the diameter of thenanoparticles formed.

FIGS. 3A, 3B, 3C, and 3D show the particle size distribution for sets ofnanoparticles formed that encapsulate various water soluble agents.

FIG. 4 shows particle size distributions for uncoated particles inchloroform (CHCl₃), uncoated particles in methanol (MeOH), and coatedparticles in water.

DETAILED DESCRIPTION

Embodiments of the invention are discussed in detail below. Indescribing embodiments, specific terminology is employed for the sake ofclarity. However, the invention is not intended to be limited to thespecific terminology so selected. A person skilled in the relevant artwill recognize that other equivalent parts can be employed and othermethods developed without parting from the spirit and scope of theinvention. All references cited herein are incorporated by reference asif each had been individually incorporated.

Encapsulation and delivery of soluble therapeutics and biologics,including peptides, proteins, DNA, and RNA, is challenging. Biologicscan exhibit poor stability, fast clearance times, immune recognition,and high costs. Nanoparticles, microparticles, and larger monolithscapable of releasing soluble therapeutics in a controlled manner thatwill protect them from degradation, clearance, and immune recognitionare desired. Biologics are presently commonly delivered via injection,thus controlled release may reduce the frequency of drug administrationand increase patient compliance.

It has been possible to make nanoparticles by rapid precipitationprocesses, described in U.S. Pat. No. 8,137,699 B2. However, theseprevious examples involved hydrophobic core materials that precipitatedout of an aqueous phase upon mixing. It was unexpected that the processcould be completely inverted and that a water soluble compound could beprecipitated into a hydrophobic solution. It was unexpected thatcopolymers could be used in a reverse way in which the polar componentis oriented inside the core of the particle and the hydrophobic lesspolar component is oriented into the less polar solution phase withstable particles resulting.

In this specification, the terms “nanoparticles”, “particles”, and“nanocarriers” are used interchangeably, unless a distinction isindicated by the context. Particles according to the invention that havehydrophilic or more polar cores are at times referred to as “inverseparticles”, to contrast them with particles that have hydrophobic orless polar cores. However, for the sake of brevity, when the contextindicates that particles having hydrophilic or more polar coresaccording to the invention are being discussed, these may be simplyreferred to as “particles” or “nanoparticles”.

The development of injectable polymeric depots for the prolonged releaseof biologics is a complex engineering challenge of optimizing biologicstability, encapsulation efficiency, loading, and release profile, aswell as ease and scalability of production. That is, the delivery systemfor a biologic should protect, prolong the release of, reduce clearancetimes of, and reduce the frequency of administration of the biologic, aswell as target the tissue(s) of interest. The aqueous processingconditions and lack of hydrophobic interfaces in hydrogel based deliverysystems make them suitable for maintaining protein stability. However,the diffusion controlled release from these systems makes it difficultto produce degradable injectable gels capable of releasing biologicsover periods of months or longer. Hydrophobic scaffolds are adept atmore prolonged release, but the formulation methods can be too harsh formost large proteins. In the double emulsion method for formingnanocarriers, it is difficult to increase loading, encapsulationefficiency, protein stability, or optimize the release profile withoutnegatively affecting another parameter. It is important to understandthese failings in order to design new formulation methods. For example,newly investigated approaches of post-loading porous PLGA capitalize onfundamental research into the closure of pores on the microparticlesurface during burst release as well as previous observations thatprotein desorption from the PLGA walls plays an important role incontrolling the release rate. Ultimately, protein and peptidetherapeutics cover a broad range of molecules, each with particularphysical characteristics and processing needs; it is unlikely that asingle polymeric depot will be suitable for the delivery of everybiologic.

The delivery of agents such as protein and peptide therapeutics or otherbiologics from polymeric systems can be through release from a monolith(including erodible implantable devices), or release from micro ornanoparticles. Monoliths include erodible implantable devices. Micro andnanoparticles may be delivered systemically or in a local depot.

Release of therapeutics from polymeric systems may be controlled in oneof two ways. In the first method, the therapeutic is conjugated to thepolymeric material of the scaffold. The therapeutic is released when itis cleaved from the scaffold. This is most commonly done with hydrogels.Because conjugation entails the formation of new chemical bonds, thesystem is subject to more rigorous FDA approval and is thus generallyundesirable. In the second method, the soluble therapeutics areencapsulated within an insoluble but erodible matrix. The erodiblematrices are hydrophobic and must be processed with hydrophobic organicsolvents. This method may be preferable because there is no chemicalmodification to the therapeutic.

Soluble materials can be encapsulated without chemical modificationthrough either (1) mixing the material directly with a scaffold material(example: PLGA) in an organic solvent or (2) forming an emulsion. Inmethod (1), the hydrophilic material often aggregates in the organicsolvent. As the solvent is removed, even at low loadings, theseaggregates produce percolating pathways resulting in an unfavorableburst release of encapsulated material. In order to improve the releaseprofile, process (2) can be used. The soluble material is contained inan aqueous phase that is encapsulated in an outer, nonmiscible, organicsolvent containing a hydrophobic scaffold material. Percolation isprevented and the emulsion is stabilized through the use of smallmolecule or polymeric surfactants. The emulsion process is completed inbatches, which is not optimal for large scale production. Additionally,the high shear rates involved in the emulsification process may denatureproteins and cleave DNA.

Flash NanoPrecipitation (FNP) is a previously patented process (U.S.Pat. No. 8,137,699 (herein, “'699 patent”), herein incorporated byreference in its entirety) to make nanoparticles with a hydrophobic coreand hydrophilic stabilizing shell (Johnson, B. K., et al., AIChE Journal(2003) 49:2264-2282). This process allows for the high loading ofhydrophobic material and can reproducibly produce particles ranging insize from the micelle size of the stabilizing material up to severalhundred nanometers. Currently, the use of FNP has been limited to theencapsulation of core material with high logP values (hydrophobic).Flash NanoPrecipitation technology can encapsulating biologics with highencapsulation efficiency and loadings greater than 75 wt %.

In a method according to the invention, polymer protected core shellnanoparticles are made by rapid precipitation, so that the resultingparticles contain hydrophilic material in their core, and anorganic-solvent soluble (less hydrophilic) shell. These nanoparticleshaving a hydrophilic core and a less hydrophilic shell can be termed“inverse” nanoparticles, in contrast with the nanoparticles of the '699patent having a hydrophobic core and a hydrophilic shell.

These “inverse” particles may be processed by covalently or non-covalentstabilizing the particles, adding a second coating of stabilizingmaterial (layer by layer FNP), and/or by incorporating them into largermonoliths or microparticles.

Nanoparticle Formation Flash NanoPrecipitation Process

The Flash NanoPrecipitation process can be used to create “inverse”particles with hydrophilic cores and/or with encapsulated water solubleagents, such as hydrophilic peptides. The process is illustrated in FIG.1 . A copolymer 102 can be dissolved in a polar process solvent at aconcentration of at least 0.1% by weight, but preferably theconcentration of copolymer is at least 0.2% by weight to form a firstprocess solution. In an embodiment, the copolymer can be dissolved inthe polar process solvent at a concentration in a range of from about0.1 wt %, 0.2 wt %, 0.5 wt %, 1 wt %, 2 wt %, 5 wt %, 10 wt %, or 20 wt% to about 0.2 wt %, 0.5 wt %, 1 wt %, 2 wt %, 5 wt %, 10 wt %, 20 wt %,or 40 wt %. A person of skill in the art will appreciate that a factorsuch as the economics of a process can limit the constrain a lower boundof concentration, and that factors such as the viscosity of the processsolution or the solubility limit of the copolymer in the polar processsolvent can constrain an upper bound of concentration. For example, ifthe viscosity of the first process solution is much greater than that ofthe nonprocess solvent, mixing of the first process solution with thenonprocess solvent may be inhibited. A person of skill in the art willappreciate that factors such as the molecular weight of the copolymerand the composition of the copolymer can affect the maximumconcentration that can be attained in the polymer solution before theviscosity becomes too high.

Examples of copolymers include but are not limited to block copolymers,graft copolymers, and random copolymers that contain regions withdifferent solvent solubilities within the same copolymer. For example, apoly(n-butyl acrylate)-block-poly(acrylic acid) (PBA-b-PAA) diblockcopolymer can be used. Examples of process solvents include, but are notlimited to, water, alcohols, acetone, acetonitrile, dimethyl sulfoxide,dimethylformamide, and mixtures thereof. The process solvent can beheated or pressurized or both to facilitate dissolution of thecopolymer, depending on the dissolution characteristics of the copolymerin the solvent.

Upon micromixing 103 the process solvent containing the copolymer with aless polar non-process solvent, the dissimilar solubilitycharacteristics of regions or portions of the copolymer are manifestedand the more polar portions of the copolymer can no longer exist in thesoluble state, so that an “inverse” nanoparticle 201 precipitates.

In an embodiment, additive water soluble target molecules 101, forexample, a hydrophilic peptide, can be added to the copolymer 102 in theprocess solvent. Upon creation of nanoparticles 201 with the copolymer,the additive target molecule 101 will be incorporated in thenanoparticle. Additive target molecules 101 that are poorly soluble inthe non-process solvent are coated, encapsulated, or confined as aparticulate core and sterically stabilized by the protective colloid ofthe copolymer 102. The nanoparticles maintain a small and stable size inthe nonprocess solvent.

In another embodiment (not shown in FIG. 1 ), the target material andcopolymer are dissolved in separate process solvent streams. The processsolvent used to dissolve the copolymer and target material may be, butare not required to be, the same. These streams are simultaneously mixedwith the non-process solvent. In another embodiment, the target materialand copolymer are dissolved in a single process solvent stream. Thisstream is then rapidly mixed with a nonprocess solvent.

The intense micromixing 103 of the process solution and the non-processsolvent can be effected in any number of geometries. The essential ideais that high velocity inlet streams cause turbulent flow and mixing thatoccurs in a central cavity. The time for process solvent/non-processsolvent mixing is more rapid than the assembly time of thenanoparticles. While not meant to be limiting, two such geometries havebeen previously described and analyzed: the Confined Impinging Jet mixer(CIJ) (Johnson, B. K., Prud'homme, R. K. Chemical processing andmicromixing in confined impinging jets. AIChE Journal 2003, 49,2264-2282; Liu, Y., Fox, R. O. CFD predictions for chemical processingin a confined impinging-jets reactor. AIChE Journal 2006, 52, 731-744)or a multi-inlet vortex mixer (MIVM) (Liu, Y., Cheng, C., Liu, Y.,Prud'homme, R. K., Fox, R. O. Mixing in a multi-inlet vortex mixer(MIVM) for flash nano-precipitation. Chemical Engineering Science 2008,63, 2829-2842). These examples are meant to be illustrative rather thanlimiting or exhaustive.

The fast mixing and high energy dissipation involved in this processprovide mixing timescales that are shorter than the timescale fornucleation and growth of particles, which leads to the formation ofnanoparticles with active agent loading contents and size distributionsnot provided by other technologies. When forming the nanoparticles viaFlash NanoPrecipitation, mixing occurs fast enough to allow highsupersaturation levels, for example, as high as 10,000, of allcomponents to be reached prior to the onset of aggregation. Thesupersaturation level is the ratio of the actual concentration of amaterial, for example, a copolymer, in a solvent to the saturationconcentration of that material in that solvent. For example, thesupersaturation levels can be at least greater than about 1, 3, 10, 30,100, 300, 1000, or 3000 and can be at most about 3, 10, 30, 100, 300,1000, 3000, or 10,000. The timescale of aggregation of the targetmaterial and copolymer self-assembly are balanced. Therefore, the targetmaterial and polymers precipitate simultaneously, and overcome thelimitations of low active agent incorporations and aggregation foundwith the widely used techniques based on slow solvent exchange (e.g.,dialysis). The Flash NanoPrecipitation process is insensitive to thechemical specificity of the components, making it a universalnanoparticle formation technique.

The size of the resulting nanoparticles from this process can becontrolled by controlling the mixing velocity used to create them, thetotal mass concentration of the copolymer and target molecules in theprocess solvent, the process and non-process solvents, the ratio of thecopolymer and target molecule, and the supersaturation of the targetmolecule and non-soluble portion of the copolymer upon mixing with thenon-process solvent. That is, there are a number of “handles” that canbe used to control the size of the nanoparticles. For example, in theformation of particles including lysozyme as the target molecule, thenanoparticle diameter can be varied with the total mass concentration asshown in FIG. 2A, with the mass percent of the target molecule lysozyme(mass percent of lysozyme with respect to lysozyme plus copolymer) asshown in FIG. 2B, with the volume fraction of acetone (fraction ofacetone with respect to acetone plus chloroform) in the nonprocesssolvent (anti solvent), the acetone being a poorer solvent for thehydrophilic lysozyme than the chloroform, as shown in FIG. 2C, and withthe volume fraction of water (H₂O) in dimethylsulfoxide (DMSO) in thepolar process solvent as shown in FIG. 2D. The experimental conditionsunder which this information was obtained is shown in Table 1, below.

TABLE 1 Agent, Effect Studied, Nonprocess & FIG. Process Stream StreamBath Lysozyme, 500 uL DMSO, 500 uL CHCl₃ 4.5 mL Effect of loadinglysozyme and polymer CHCl₃ on nanoparticle at varying ratios, (NP) size,(lysozyme + FIG. 2B polymer) = 10 mg/mL Lysozyme, 500 uL DMSO, 500 uL4.5 mL Nonprocess 5 mg/mL lysozyme, antisolvent antisolvent solventeffect, 5 mg/mL polymer mixture mixture FIG. 2C Lysozyme, 500 uL DMSO,500 uL CHCl₃ 4.5 mL Total mass equal masses of CHCl₃ concentration,lysozyme and polymer FIG. 2A Lysozyme, 500 uL DMSO with 500 uL CHCl₃ 4.5mL Water effect, set vol % MQ, CHCl₃ FIG. 2D 5 mg/mL lysozyme, 5 mg/mLpolymer

Without being bound by theory, for example, as the total massconcentration is increased (FIG. 2A), the protein aggregation rate cangrow more quickly than the nucleation rate of polymer self assembly. Atlower mass percentages of lysozyme, as the mass percentage of lysozymeis increased (FIG. 2B), the size initially decreases. Without beingbound by theory, this may be because of a faster nucleation rate, i.e.,with more nucleation sites there are more, but smaller, particles.Alternatively, this may be because of better packing of the PAA blocks,i.e., the negative charges of the PAA repel, and water and/or other corematerials can shield this effect. At higher mass percentages oflysozyme, as the mass percentage of lysozyme is increased, the sizeincreases. Without being bound by theory, this may be because at highpercentages, large particles form because they have a higher volume tosurface area ratio than small particles. As the volume fraction ofacetone in the nonprocess solvent (antisolvent) is increased (FIG. 2C),the nonprocess solvent becomes less polar. At lower acetoneconcentrations, as the acetone concentration is increased, the diameterof the particles formed decreases, because the nucleation rate of thecopolymer increases and this dominates the size of the particles formed,i.e., with more nucleation sites more, but smaller, particles areformed. At higher acetone concentrations, as the acetone concentrationis increased, the rate of aggregation of the lysozyme protein isenhanced and this dominates the size of the particles formed. As thevolume fraction of water is increased (FIG. 2D), the process solventbecomes more polar. At lower water concentrations, as the waterconcentration is increased, the water acts as a nucleation site, helpingthe core of the nanoparticle to pack more tightly, so that the size ofthe nanoparticles decreases. At higher water concentrations, as thewater concentration is increased, the water becomes integrated into thecore, so that the size of the nanoparticles increases.

Nanoparticles as small as 40 nm diameter can be obtained at 50% loading(loading being the percentage of water soluble agent with respect to thewater soluble agent plus copolymer). Stable nanoparticles can beobtained with 75% loading, although the diameter of the nanoparticlesbecame too large to measure using dynamic light scattering (DSL).

Nanoparticles can be produced from copolymers that are dissolved in aprocess solvent with no target material added.

Using the methods according to the invention, particles can be made thathave sizes in the range of 15 nm to 10500 nm, sizes in the range of 20nm to 6000 nm, sizes in the range of 20 nm to 1000 nm, sizes in therange of 35 nm to 400 nm, or sizes in the range of 40 nm to 300 nm.Sizes can be determined by dynamic light scattering. For example,particles can be made that have sizes of at least about 15 nm, 20 nm, 35nm, 40 nm, 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 600 nm, 1000 nm, 2000nm, 4000 nm, or 6000 nm, and have sizes of at most about 20 nm, 35 nm,40 nm, 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 600 nm, 1000 nm, 2000 nm,4000 nm, 6000 nm, or 10500 nm. Sizes reported and cited herein are theintensity average reported values as determined by the Malvern Nanosizerdeconvolution program. Other intensity weighted deconvolution methodscan be used to determine sizes of the nanoparticles.

Encapsulated Material

Encapsulated material (target molecules) must be sufficiently polar thatit rapidly precipitates in the less polar non-process solvent. Moleculesthat do not meet these criteria may be chemically modified to increasetheir water solubility and propensity to precipitate in the organicnon-process solvent. Examples of biologic material that may beencapsulated include, but are not limited to, peptides, proteins, DNA,RNA, saccharides, and derivatives, conjugates, and/or analogs thereof.For example, glucagon-like peptide-1 (GLP-1) may be encapsulated. Smallmolecule water soluble therapeutics and imaging agents may also beencapsulated. Soluble stabilizing agents may be encapsulated inparticles to provide stability to the particle for its use or forsubsequent processing steps. Any of these materials may also beco-precipitated within a single particle. Hydrophilic material may beencapsulated for the sole purpose of adding stability to the particlesduring post processing. For example, material with molecular weightsbetween 100 and 10,000,000 Daltons (Da) may be encapsulated. Materialwith molecular weights between 250 and 10,000,000 Da may beencapsulated. Material with molecular weights between 100 and 1,000,000Da may be encapsulated. Material with molecular weights between 250 and1,000,000 Da may be encapsulated. Material with molecular weightsbetween 100 and 200,000 Da may be encapsulated.

Certain encapsulated materials may be multifunctional. For example,tobramycin is cationic and can itself be crosslinked with a copolymer.

The encapsulated material may be incorporated into the particle at arange of loadings. For example, the mass of the encapsulated materialmay be greater than or equal to the mass of the copolymer. For example,the concentration of the encapsulated material in the first processsolution may be from about 0.1 wt %, 0.2 wt %, 0.5 wt %, 1 wt %, 2 wt %,5 wt %, 10 wt %, or 20 wt % to about 0.2 wt %, 0.5 wt %, 1 wt %, 2 wt %,5 wt %, 10 wt %, 20 wt %, or 40 wt %.

Solvents

Formation of nanoparticles requires a process solvent and non-processsolvent stream. The process and non-process solvents may be a pure (thatis, a single) liquid compound or a mixture of two or more pure liquidcompounds. Other non-liquid compounds that aid in the solvent quality ofthe streams may be added and are also considered part of the solvent.These excipient compounds may or may not be in the final nanoparticle ormicroparticle construct, depending on the requirements of the finalproduct.

The polar process solvent containing the copolymer is chosen such thatthe copolymer is molecularly dissolved. This requires that the processsolvent solubilize all parts of the copolymer. The process solventcontaining the material to be encapsulated, if present, is also chosensuch that material is molecularly dissolved. These process solvents maybe, but are not required to be, the same. In some cases, both thecopolymer and material to be encapsulated may be dissolved in a singlesolution of the process solvent. In order to dissolve the water solublematerial to be encapsulated, the process solvent is more polar than thenon-process solvent. Examples of process solvents include, but are notlimited to, water, alcohols, methanol, ethanol, dimethyl formamide(DMF), dimethyl sulfoxide (DMSO), acetonitrile, acetone, N-methylpyrrolidone (NMP), and mixtures thereof. Acids, bases, and salts are afew examples of additives that may be used to aid in the solubilizationof the copolymer and encapsulated material in the process solvent.

The solutions of process solvent containing copolymer and material to beencapsulated are mixed with a nonprocess solvent. The non-processsolvent must be capable of changing the local molecular environment ofthe copolymer and causing local precipitation of the more polar sectionsof the polymer. The nonprocess solvent is chosen such that the morepolar sections of the copolymer rapidly precipitate and the morenonpolar (less polar) sections of the copolymer remain solubilized.Thus, the copolymer will self-assemble into micelles or other structuresin the nonprocess solvent. The nonprocess solvent is chosen such thatthe target material to be encapsulated rapidly precipitates in the finalmixture. In most cases it is preferable for the process and non-processsolvents to be fully miscible at the final composition. In some cases,no more than 20 volume percent of the process solvent may phase separatein the final composition. In general, this is only acceptable if thephase separated solvent goes to the core of the particles and there isno macroscopic separation. Nonprocess solvents include, but are notlimited to, chloroform, dichloromethane, alkanes such as hexane, etherssuch as diethyl ether, tetrahydrofuran (THF), toluene, acetone, andmixtures thereof. Acids, bases, and salts are a few examples ofadditives that may be used to aid in the precipitation of theencapsulated material and sections of the copolymer. Solvent choices aremade based on the solubilities of the copolymer and encapsulatedmaterials. It is important to note that process solvents of one systemmay work well as the nonprocess solvent in another system, thus theexamples given above for process and nonprocess solvents should not beconsidered distinct.

Copolymers

The stabilizing polymer can be a copolymer of a more polar block coupledwith a more nonpolar (less polar) block. The term “block” may beinterpreted as either a distinct domain with a single molecularcomposition, or it may mean a region of the polymer chain which hasregions that are predominantly more polar and other regions that areless polar. The polarity may be imparted by the monomers comprising thepolymer backbone or grafted pendant groups or chains attached to themain polymer backbone. For example, the copolymer may be amphiphilic(the more nonpolar block is not water soluble), however, this is not arequirement and copolymers may be fully water soluble or fully non-watersoluble, as long as solubilities of the blocks differ significantlyenough in the nonprocess solvent. The copolymer should self-assemble inthe nonprocess solvent, with the more polar blocks precipitating and themore nonpolar blocks remaining soluble. When used in the FNP process tomake particles, the more polar blocks go to the core of the particle,and the more nonpolar blocks form a sterically protective shell. Thesterically protective shell prevents particle aggregation and preventspercolation of encapsulated material during post processing steps.

Nanoparticles formed by the disclosed process can be formed with graft,block, or random copolymers. For example, these copolymers can have amolecular weight between about 1000 g/mole and about 1,000,000 g/mole,or between about 3000 g/mole and about 25,000 g/mole, or at least about2000 g/mole.

The copolymers are comprised of repeat units or blocks that havedifferent solubility characteristics. Typically, these repeat units arein groups of at least two comprising a block of a given character.Depending on the method of synthesis, these blocks could be of all thesame repeat unit or contain different repeat units dispersed throughoutthe block, but still yielding blocks of the copolymer with polar andmore non-polar portions. These blocks can be arranged into a series oftwo blocks (diblock) or three block (triblock), or more (multiblock),forming the backbone of a block copolymer. In addition, the polymerchain can have chemical moieties covalently attached or grafted to thebackbone. Such polymers are graft polymers. Block units making up thecopolymer can occur in regular intervals or they can occur randomlymaking a random copolymer. In addition, grafted side chains can occur atregular intervals along the polymer backbone or randomly making arandomly grafted copolymer. In graft polymers, polar blocks may begrafted on a non-polar polymer. More commonly, non-polar blocks aregrafted on a more polar polymer chain. In graft copolymers, the lengthof a grafted moiety can vary. Preferably, the grafted segments areequivalent to 2 to 22 ethylene units in length. In addition, thegrafting of the polymer backbone can be useful to enhance solvation ornanoparticle stabilization properties.

The copolymer used in the compositions and methods of the invention maybe comprised of blocks of at least two repeat units or with a minimumcontour length the equivalent of at least 25 ethylene units. Contourlengths are the linear sum of the polymer backbone, the moleculardimensions of which can be approximated using the Polymer Handbook, 4thEdition, eds. J. Brandrup, E. H. Immergut, and E. A. Grulke, assoc. ed.A. Abe, D. R. Bloch, 1999, New York, John Wiley & Sons, which is herebyincorporated by reference in its entirety.

Examples of suitable nonpolar blocks in a copolymer include but are notlimited to the following: acrylates including methyl acrylate, ethylacrylate, propyl acrylate, n-butyl acrylate (BA), isobutyl acrylate,2-ethyl acrylate, and t-butyl acrylate; methacrylates including ethylmethacrylate, n-butyl methacrylate, and isobutyl methacrylate;acrylonitriles; methacrylonitrile; vinyls including vinyl acetate,vinylversatate, vinylpropionate, vinylformamide, vinylacetamide,vinylpyridines, vinyl phenols and vinyllimidazole; aminoalkyls includingaminoalkylacrylates, aminoalkylsmethacrylates, andaminoalkyl(meth)acrylamides; styrenes; cellulose acetate phthalate,cellulose acetate succinate, hydroxypropylmethylcellulose phthalate,poly(D,L-lactide), poly (D,L-lactide-co-glycolide), poly(glycolide),poly(hydroxybutyrate), poly(alkylcarbonate) and poly(orthoesters),polyesters, poly(hydroxyvaleric acid), polydioxanone, poly(ethyleneterephthalate), poly(malic acid), poly(tartronic acid), polyanhydrides,polyphosphazenes, poly(amino acids), lactic acid, caprolactone, glycolicacid, and their copolymers (see generally, Illum, L., Davids, S. S.(eds.) Polymers in Controlled Drug Delivery Wright, Bristol, 1987;Arshady, J. Controlled Release 17:1-22, 1991; Pitt, Int. J. Phar.59:173-196, 1990; Holland et al., J. Controlled Release 4:155-0180,1986); hydrophobic peptide-based polymers and copolymers based onpoly(L-amino acids) (Lavasanifar, A., it al., Advanced Drug DeliveryReviews (2002) 54:169-190), poly(ethylene-vinyl acetate) (“EVA”)copolymers, silicone rubber, polyethylene, polypropylene, polydienes(polybutadiene, polyisoprene and hydrogenated forms of these polymers),maleic anhydride copolymers of vinyl methylether and other vinyl ethers,polyamides (nylon 6,6), polyurethane, poly(ester urethanes), poly(etherurethanes), poly(esterurea). For example, polymeric blocks can includepoly(ethylenevinyl acetate), poly(D,L-lactic acid) oligomers andpolymers, poly(L-lactic acid) oligomers and polymers, poly(glycolicacid), copolymers of lactic acid and glycolic acid, poly(caprolactone),poly(valerolactone), polyanhydrides, copolymers of poly(caprolactone) orpoly(lactic acid) For non-biologically related applications polymericblocks can include, for example, polystyrene, polyacrylates, andbutadienes.

Natural products with sufficient hydrophobicity to act as the non-polarportion of the polymer include: hydrophobic vitamins (for examplevitamin E, vitamin K, and A), carotenoids, and retinols (for example,beta carotene, astaxanthin, trans and cis retinal, retinoic acid, folicacid, dihydrofolate, retinylacetate, retinyl palmintate),cholecalciferol, calcitriol, hydroxycholecalciferol, ergocalciferol,alpha-tocopherol, alpha-tocopherol acetate, alphatocopherol nicotinate,and estradiol. For example, a natural product is vitamin E which can bereadily obtained as a vitamin E succinate, which facilitatesfunctionalization to amines and hydroxyls on the active species.

Examples of suitable polar blocks in an amphiphilic polymer that is ablock copolymer include, but are not limited to the following:carboxylic acids including acrylic acid, methacrylic acid, itaconicacid, and maleic acid; polyoxyethylenes or poly ethylene oxide;polyacrylamides and copolymers thereof withdimethylaminoethylmethacrylate, diallyldimethylammonium chloride,vinylbenzylthrimethylammonium chloride, acrylic acid, methacrylic acid,2-crrylamideo-2-methylpropane sulfonic acid and styrene sulfonate,polyvincyl pyrrolidone, starches and starch derivatives, dextran anddextran derivatives; polypeptides, such as polylysines, polyarginines,polyaspartic acids, polyglutamic acids; poly hyaluronic acids, alginicacids, polylactides, polyethyleneimines, polyionenes, polyacrylic acids,and polyiminocarboxylates, gelatin, and unsaturated ethylenic mono ordicarboxylic acids. To prepare anionic copolymers, acrylic acid,methacrylic acid, and/or poly aspartic acid polymers can be used. Toproduce cationic copolymers, DMAEMA (dimethyl aminoethylmethacrylate),polyvinyl pyridine (PVP), and/or dimethylaminoethylacrylamide (DMAMAM)can be used. A listing of suitable polar, water soluble, polymers can befound in Handbook of Water-Soluble Gums and Resins, R. Davidson,McGraw-Hill (1980).

The lists above of nonpolar and polar polymers should not be consideredexclusive of one another. Copolymers of two polymers given in a singlelist may have sufficient differences in solubilities in a givennonprocess solvent to be used in this process. As an illustrativeexample, poly(ethylene oxide) and poly(acrylic acid) are both given inthe list of polar polymers. However, poly(ethylene oxide) is soluble inchloroform and acetone, while poly(acrylic acid) is not. Therefore,copolymers of poly(ethylene oxide) and poly(acrylic acid) may be used inthis process with chloroform or acetone as the nonprocess solvent.

Nanoparticle Processing Particle Stabilization

The particles are formed and stable in the organic nonprocess solvent.In most applications, it is required that the final construct be stablein aqueous environments for a set, nonnegligible amount of time. Inorder to process the particles into an aqueous environment, particlestabilization is required. Without stabilization, the particle maydissolve, aggregate, and/or release the water soluble target materialfrom the core.

In an embodiment according to the invention, sections of the core of theparticle may be stabilized The core refers to the more polar sections ofthe copolymer and encapsulated material. Material may be incorporatedinto the core specifically for the purpose of particle stabilization.For example, the portions of the copolymer in the core may becrosslinked 203 to form a particle with a crosslinked core 301. Inanother embodiment, the shell of the particle may be stabilized. Theshell refers to the more nonpolar sections of the copolymer that aresoluble in the nonprocess solvent.

Stabilization can involve the formation of new covalent bonds. Forexample, the copolymer of the core (and, in some cases, the encapsulatedmaterial) of the particle may be cross-linked through the formation ofnew covalent bonds. The bonds may be formed directly between groups onthe copolymer. Covalent bonds may be formed by adding a crosslinkingmaterial to the core for the specific purpose of cross-linking thepolymer in the core. The crosslinking material (stabilizing material)may be added to the core of the particle during the FNP process. Forexample, the crosslinking material can be included in the processsolvent. As another example, the crosslinking material can be includedin the nonprocess solvent.

Alternatively, the crosslinking material may be added to the solutionafter the particle has formed. For example, the particle may be“incubated” with a crosslinking material, such as a metal salt, and thecrosslinking material may interact with a more polar portion of thecopolymer, e.g., PAA, for example, through ionic and/or chelationeffects. The degree of crosslinking realized can then be characterizedby suspending the particle in a good solvent for the more polar portionof the copolymer. Particles with tight (dense) crosslinking can exhibitminimal swelling and can be associated with high levels of metalpartitioning into the hydrophilic core and strong metal interactionswith the more polar part of the polymer. Particles with loosecrosslinking can exhibit high levels of swelling and can be associatedwith low levels of metal partitioning into the hydrophilic core and weakmetal interactions with the more polar part of the polymer. If thepartitioning of the metal into the core is very low and or theinteraction of the metal with the more polar part of the polymer is veryweak, then the particle may disassemble and dissolve in the solvent.

If the crosslinking material is added after the particles have beenformed, the crosslinking may be diffusion limited and only occur on theouter layers of the core. If the crosslinking material is added to thesolution after the particles have been formed, the particle may becross-linked throughout the core if the core is swollen with solvent orif the cross-linking material is small enough to diffuse throughout thecore. The shell of the particle may be cross-linked through theformation of new covalent bonds. The bonds may be formed directlybetween groups on the copolymer, or through the addition of an extracrosslinking material.

Examples of covalent chemistries that may be used include, but are notlimited to carbodiimide coupling of carboxylic acids to alcohols orcarboxylic acids to amines, the coupling of activated esters to alcoholsor amines, maleimide-thiol chemistry, Micheal addition, azidealkyne“click” chemistry, UV or light activated chemistries, and/or disulfideformation.

Stabilization can be obtained through non-covalent interactions. Thecore of the particle may be cross-linked through non-covalentinteractions. The interactions may be directly between groups on thecopolymer. Non-covalent interactions may be formed by adding acrosslinking material to the core for the specific purpose ofcross-linking the polymer in the core. This crosslinking material may beadded to the core of the particle during the FNP process. Alternatively,this crosslinking material may be added to the solution after theparticle has formed. If the crosslinking material is added after theparticles have been formed, the crosslinking may be diffusion limitedand only occur on the outer layers of the core. If the crosslinkingmaterial is added to the solution after the particles have been formed,the particle may be crosslinked throughout the core if the core isswollen with solvent or if the crosslinking material is small enough todiffuse throughout the core. The shell of the particle may becross-linked through noncovalent interactions. The interactions may beformed directly between groups on the copolymer, or through the additionof an extra crosslinking material.

Examples of non-covalent interactions that may be used include, but arenot limited to, ionic interactions, acid-base interactions, metalchelation, interactions between polyhistidines and a metal such asnickel, and/or strong hydrogen bonding. An example of non-covalentparticle stabilization is the use of Cr(III) to stabilize thepoly(acrylic acid) core of a nanoparticle. For example, chromium (III)acetate and/or chromium (III) bromide can be used as crosslinkingmaterials. The crosslinking may proceed through ligand exchange. Thesolvents used can act as ligands. For example, the interaction of thecation in a crosslinking salt should be stronger with the more polarportion of the copolymer to be crosslinked in the core than with theanion in the salt.

Other crosslinking materials (crosslinking agents) that can be used toinduce non-covalent crosslinking include alkaline earth halides,magnesium halides, calcium halides, metal halides, transition metalhalides, and iron halides. Metal salts can be used. Additionalcrosslinking materials that can be used are metal acetates, alkalineearth acetates, transition metal acetates, and calcium acetate. Thecrosslinking ability of a given cation (e.g., a metal) depends on theaccompanying anion. The crosslinking ability of a crosslinking material,e.g., a salt, can depend on the process solvent and nonprocess solventused. A crosslinking material can include a metal that is biologicalinteresting or functional or otherwise useful. For example, Fe(III),Ca(II), and Zn(II) cations are biocompatible. (gadolinium(III)) isactive in magnetic resonance imaging (MRI), and, therefore, can beuseful as a tracer.

Some crosslinking materials that work well when conducting crosslinkingduring nanoparticle formation, e.g., during the FNP process, includepolyamines, such as spermine, and certain chloride salts, such asmagnesium chloride, calcium chloride, and iron(III) chloride. Forexample, such crosslinking materials can be used with PBA-b-PAAcopolymer, methanol, dimethylsulfoxide, and/or water as the processsolvent, and acetone and/or chloroform as the nonprocess solvent. It maybe necessary to include some water in the process solvent for thecrosslinking to occur. In some systems, calcium chloride, magnesiumchloride, and spermine may act as weak crosslinkers. An iron(III) salt,such as iron(III) chloride, may induce strong crosslinking.

Multiple types of stabilization chemistries may be employed within agiven particle. Stabilization may occur in the core, in the shell, atthe interface, or in multiple locations within a given particle.

For many applications, particle degradation and release of encapsulatedmaterial is required. The type of stabilization chemistry used, and thedensity of the crosslinked network, may affect the degradation kineticsof the particle. The type of stabilization chemistry used, and thedensity of the cross-linked network, may also or alternatively affectthe release kinetics of encapsulated material from the core of theparticle.

For some applications, it is required that the encapsulated material isnot chemically modified. In these cases, non-covalent interactionsshould be used to stabilize the particle. However, covalent crosslinkingmay be used as long as the chemistry is specific to the copolymer anddoes not modify the encapsulated material.

After crosslinking, if the more nonpolar blocks of the copolymer arewater soluble, the particles may be placed directly in an aqueousenvironment. The particle shell will provide steric stabilization. Anexample is particles composed of poly(acrylic acid)-b-poly(ethyleneoxide) that are formed with chloroform as the non-process solvent anduse Cr(III) to crosslink the poly(acrylic acid) core. Once the particleshave been crosslinked they may be placed in an aqueous environment, andthe poly(ethylene oxide) will provide steric stabilization and preventparticle aggregation.

After crosslinking, if the more non-polar blocks of the copolymer areshort and the core of the particle is charged, the particles may beplaced in an aqueous environment. This requires that the core swellsufficiently in the aqueous environment such that charged patches of thecore are no longer protected by the nonpolar blocks of the copolymer.These patches must be sufficiently large and charged in order for theparticles to not aggregate (charge stabilized).

Particle Coating—Layer by Layer Flash NanoPrecipitation

After particle stabilization, a second layer of an amphiphilic polymer302 may be coated onto 303 the surface of the particle 301 to form ananoparticle having a coating 401 (see FIG. 1 ). This second layer ofamphiphilic polymer 302 can be referred to as the “coating”. Thiscoating may be done to modify the surface properties of the particle tomake it stable in an aqueous environment. For example, this may beuseful if the shell of the particle—that is, the more nonpolar sectionsof the copolymer—is not water soluble. Particle coating with astabilizing amphiphilic polymer 302 may be accomplished through a secondFlash NanoPrecipitation step 303. The particles 301 must be sufficientlystabilized prior to being coated to be able to withstand the coatingprocess.

The stabilizing amphiphilic polymer 302 can be a copolymer of ahydrophilic block coupled with a hydrophobic block. Nanoparticles coatedby the disclosed process can be coated with graft, block, or randomamphiphilic copolymers. These amphiphilic polymers can have a molecularweight of between about 1000 g/mole and about 50,000 g/mole, betweenabout 3000 g/mole and about 25,000 g/mole, or at least 2000 g/mole.Examples of suitable hydrophobic blocks in an amphiphilic polymer thatis a block copolymer include, but are not limited to the following:acrylates including methyl acrylate, ethyl acrylate, propyl acrylate,n-butyl acrylate (BA), isobutyl acrylate, 2-ethyl acrylate, and t-butylacrylate; methacrylates including ethyl methacrylate, n-butylmethacrylate, and isobutyl methacrylate; acrylonitriles;methacrylonitrile; vinyls including vinyl acetate, vinylversatate,vinylpropionate, vinylformamide, vinylacetamide, vinylpyridines, vinylphenols and vinyllimidazole; aminoalkyls including aminoalkylacrylates,aminoalkylmethacrylates, and aminoalkyl(meth)acrylamides; styrenes;cellulose acetate phthalate, cellulose acetate succinate,hydroxypropylmethylcellulose phthalate, poly(D,L lactide), poly(D,L-lactide-co-glycolide), poly(glycolide), poly(hydroxybutyrate),poly(alkylcarbonate) and poly(orthoesters), polyesters,poly(hydroxyvaleric acid), polydioxanone, poly(ethylene terephthalate),poly(malic acid), poly(tartronic acid), polyanhydrides,polyphosphazenes, poly(amino acids) and their copolymers (see generally,Illum, L., Davids, S. S. (eds.) Polymers in Controlled Drug DeliveryWright, Bristol, 1987; Arshady, J. Controlled Release 17:1-22, 1991;Pitt, Int. J. Phar. 59:173-196, 1990; Holland et al., J. ControlledRelease 4:155-0180, 1986); hydrophobic peptide-based polymers andcopolymers based on poly(L-amino acids) (Lavasanifar, A., it al.,Advanced Drug Delivery Reviews (2002) 54:169-190), poly(ethylene-vinylacetate) (“EVA”) copolymers, silicone rubber, polyethylene,polypropylene, polydienes (polybutadiene, polyisoprene, and hydrogenatedforms of these polymers), maleic anhydride copolymers of vinylmethylether and other vinyl ethers, polyamides (nylon 6,6),polyurethane, poly(ester urethanes), poly(ether urethanes), andpoly(esterurea). For example, polymeric blocks includepoly(ethylenevinyl acetate), poly(D,L-lactic acid) oligomers andpolymers, poly(L-lactic acid) oligomers and polymers, poly(glycolicacid), copolymers of lactic acid and glycolic acid, poly(caprolactone),poly(valerolactone), polyanhydrides, copolymers of poly(caprolactone) orpoly(lactic acid). For example, for non-biologically relatedapplications polymeric blocks can include polystyrene, polyacrylates,and butadienes.

Natural products with sufficient hydrophobicity to act as thehydrophobic portion of the amphiphilic polymer include, for example,hydrophobic vitamins (for example, vitamin E, vitamin K, and vitamin A),carotenoids and retinols (for example beta carotene, astaxanthin, transand cis retinal, retinoic acid, folic acid, dihydrofolate,retinylacetate, retinyl palmintate), cholecalciferol, calcitriol,hydroxycholecalciferol, ergocalciferol, alpha-tocopherol,alpha-tocopherol acetate, alpha-tocopherol nicotinate, and estradiol.The preferred natural product is vitamin E which can be readily obtainedas a vitamin E succinate, which facilitates functionalization to aminesand hydroxyls on the active species.

Examples of suitable hydrophilic blocks in an amphiphilic polymerinclude but are not limited to the following: carboxylic acids includingacrylic acid, methacrylic acid, itaconic acid, and maleic acid;polyoxyethylenes or poly ethylene oxide; polyacrylamides and copolymersthereof with dimethylaminoethylmethacrylate, diallyldimethylammoniumchloride, vinylbenzylthrimethylammonium chloride, acrylic acid,methacrylic acid, 2-crrylamideo-2-methylpropane sulfonic acid andstyrene sulfonate, polyvincyl pyrrolidone, starches and starchderivatives, dextran and dextran derivatives; polypeptides, such aspolylysines, polyarginines, polyglutamic acids; poly hyaluronic acids,alginic acids, polylactides, polyethyleneimines, polyionenes,polyacrylic acids, and polyiminocarboxylates, poly(ethylene glycol),gelatin, and unsaturated ethylenic mono or dicarboxylic acids. Forexample, the hydrophilic blocks can be of poly(ethylene glycol). Forexample, the hydrophilic blocks can be of poly(ethylene oxide) and polyhydroxyl propyl acrylamide and methacrylamide to prepare neutral blockssince these materials are in currently approved medical applications. Toprepare anionic copolymers acrylic acid and methacrylic acid and polyaspartic acid polymers can be used. To produce cationic amphiphilicpolymers DMAEMA (dimethylaminoethylmethacrylate), polyvinyl pyridine(PVP) or dimethylaminoethylacrylamide (DMAMAM) can be used.

For example, the blocks can be diblock, triblock, or multiblock repeats.Preferably, block copolymers can include blocks of polystyrene,polyethylene, polybutyl acrylate, polybutyl methacrylate, polylacticacid (PLA), polyglutamic acid (PGA) and PLGA copolymers,polycaprolactone, polyacrylic acid, polyoxyethylene and polyacrylamide.A listing of suitable hydrophilic polymers can be found in Handbook ofWater-Soluble Gums and Resins, R. Davidson, McGraw-Hill (1980).

For example, the amphiphilic polymer can bepolystyrene-block-poly(ethylene glycol) (PS-b-PEG), poly(lacticacid)-block-poly(ethylene glycol) (PLA-b-PEG),poly(caprolactone)-block-poly(ethylene glycol) (PCL-b-PEG),poly(lactic-co-glycolic acid)-block-poly(ethylene glycol) (PLGA-b-PEG),or tri-block forms of the diblock copolymers listed above. Furthermore,triblock copolymers such as poly(ethylene oxide)-block-poly(propyleneoxide)-block-poly(ethylene oxide) (PEO-b-PPO-b-PEO) or poly(ethyleneoxide)-block-poly(butylene oxide)-block-poly(ethylene oxide)(PEO-b-PBO-b-PEO) may be used.

In an amphiphilic polymer that is a graft copolymer, the length of agrafted moiety can vary. For example, the grafted segments can be alkylchains of 4 to 22 carbons or equivalent to 2 to 11 ethylene units inlength. Grafted groups may also include lipids, phospholipids orcholersterol. The grafting of the polymer backbone can be useful toenhance solvation or nanoparticle stabilization properties. A graftedbutyl group on the hydrophobic backbone of a diblock copolymer of apolyethylene and polyethylene glycol should increase the solubility ofthe polyethylene block. Suitable chemical moieties grafted to the blockunit of the copolymer comprise alkyl chains containing species such asamides, imides, phenyl, carboxy, aldehyde, or alcohol groups.

The method of coating the nanoparticles is as previously described byJohnson et al., termed “Flash NanoPrecipitation” (FNP), Johnson, B. K.,et al., AIChE Journal (2003) 49:2264-2282 and U.S. Pat. No. 8,137,699,which are incorporated herein by reference in their entirety. FNP is arapid, single-step block copolymer-directed precipitation process. Theparticles can be treated to remove residual solvent, such as chloroform.The particles and amphiphilic block copolymers are dissolved in awater-miscible organic solvent. Acceptable solvents include, but are notlimited to, tetrahydrofuran (THF), dimethylformamide, acetonitrile,acetone, low molecular weight alcohols such as methanol and ethanol,dimethyl sulfoxide (DMSO), or mixtures thereof. Solvent quality israpidly reduced by micromixing against water or an aqueous buffer ormixture to produce supersaturation levels as high as 10,000 to driverapid precipitation wherein the time of mixing is faster than theaggregation of the nanoparticles and balances with the timescale ofblock copolymer self-assembly. This process is capable of producingcontrolled size, polymer stabilized, and protected nanoparticles.

The Flash NanoPrecipitation coating technique is based on amphiphilicdiblock copolymer arrested aggregation of the nanoparticles produced bythe initial FNP process. Amphiphilic diblock copolymers dissolved in agood solvent can form micelles when the solvent quality for one block isdecreased. The intense micromixing can be effected in any number ofgeometries. The essential idea is that high velocity inlet streams causeturbulent flow and mixing that occurs in a central cavity. The time forsolvent/antisolvent mixing is more rapid than the assembly time of thenanoparticles. While not meant to be limiting, two such geometries havebeen previously described and analyzed: the Confined Impinging Jet mixer(CIJ) or a multi-inlet vortex mixer (MIVM). These examples are meant tobe illustrative rather than limiting or exhaustive, and were discussedpreviously.

The vortex mixer consists of a confined volume chamber where one jetstream containing the diblock copolymer dissolved and particlessuspended in a water-miscible solvent, such as THF, acetone, or amixture, is mixed at high velocity with another jet stream containingwater, an anti-solvent for the nanoparticle shell and the hydrophobicblock of the copolymer. The fast mixing and high energy dissipationinvolved in this process provide timescales that are shorter than thetimescale for nucleation and growth of particles, which leads to theformation of nanoparticles with active agent loading contents and sizedistributions not provided by other technologies. When coating thenanoparticles via Flash NanoPrecipitation, mixing occurs fast enough toallow high supersaturation levels of all components to be reached priorto the onset of aggregation. Therefore, the particles and polymersprecipitate simultaneously, and overcome the limitations of low activeagent incorporations and aggregation found with the widely usedtechniques based on slow solvent exchange (e.g., dialysis). The FlashNanoPrecipitation process is insensitive to the chemical specificity ofthe components, making it a universal nanoparticle coating technique.

The coating formed by the amphiphilic polymer can have an inner regionand an outer region. The inner region can include hydrophobic regions ofthe amphiphilic copolymer, and the outer region can include hydrophilicregions of the amphiphilic copolymer.

The concentrations, amphiphilic polymers, and solvents used in thecoating process may be optimized such that individual particles arecoated, or particles aggregate to a desired size prior to being coated.For example, increasing the amount of amphiphilic polymer used to coatconcentrated nanoparticles relative to the amount of copolymer in theparticles themselves can result in smaller particle diameter. Whencoating concentrated nanoparticles, more than one nanoparticle can beincorporated in a stabilizing shell of amphiphilic polymer.

When coating dilute nanoparticles, the amount of amphiphilic polymerrelative to the amount of copolymer in the particles themselves may havelittle or no effect on the resultant particle diameter.

Coating the particles modifies the surface chemistry of the particles.Coating the particles may change the stability and degradation kineticsof the particles in an aqueous media. Coating the particles may changethe release kinetics of encapsulated material.

Coated nanoparticles have been formed with glutathione (GSH), lysozyme,vancomycin, or tobramycin as the encapsulated material.

Particle Incorporation into Microparticles and Monoliths

The particles may be incorporated into microparticles or largermonoliths. The hydrophobic polymer block can prevent percolation andallow for high loading of the encapsulated material, stabilizationduring processing, and controlled release.

If the particles are being incorporated into a hydrophobic scaffold thatis processed in a poor solvent for the particle core, the particle maybe adequately stabilized by the less polar polymer block prior toprocessing.

The hydrophilic active compound or biologic compound is captured in theinterior of the particle formed by the first processing step into thehydrophobic process solvent. Organic polymers soluble in the hydrophobicprocess solvent may be added to the particle dispersion. Polymers thatmight be added include biocompatible and or biodegradable polymers.Nonlimiting examples of these polymers would include: acrylatesincluding methyl acrylate, ethyl acrylate, propyl acrylate, n-butylacrylate (BA), isobutyl acrylate, 2-ethyl acrylate, and t-butylacrylate; methacrylates including ethyl methacrylate, n-butylmethacrylate, and isobutyl methacrylate and copolymers of theseacrylates; acrylonitriles; methacrylonitrile; vinyls including vinylacetate, vinylversatate, vinylpropionate, vinylformamide,vinylacetamide, vinylpyridines, vinyl phenols and vinyllimidazole;aminoalkyls including aminoalkylacrylates, aminoalkylsmethacrylates, andaminoalkyl(meth)acrylamides; styrenes; cellulose acetate phthalate,cellulose acetate succinate, hydroxypropylmethylcellulose phthalate,poly(D,L lactide), poly (D,L-lactide-co-glycolide), poly(glycolide),poly(hydroxybutyrate), poly(alkylcarbonate) and poly(orthoesters),polyesters, poly(hydroxyvaleric acid), polydioxanone, poly(ethyleneterephthalate), poly(malic acid), poly(tartronic acid), polyanhydrides,polyphosphazenes, poly(amino acids) and their copolymers (see generally,Illum, L., Davids, S. S. (eds.) Polymers in Controlled Drug DeliveryWright, Bristol, 1987; Arshady, J. Controlled Release 17:1-22, 1991;Pitt, Int. J. Phar. 59:173-196, 1990; Holland et al., J. ControlledRelease 4:155-0180, 1986); hydrophobic peptide-based polymers andcopolymers based on poly(L-amino acids) (Lavasanifar, A., it al.,Advanced Drug Delivery Reviews (2002) 54:169-190), poly(ethylene-vinylacetate) (“EVA”) copolymers, silicone rubber, polyethylene,polypropylene, polydienes (polybutadiene, polyisoprene and hydrogenatedforms of these polymers), maleic anhydride copolymers of vinylmethylether and other vinyl ethers, polyamides (nylon 6,6),polyurethane, poly(ester urethanes), poly(ether urethanes),poly(esterurea). Examples of polymeric blocks include poly(ethylenevinylacetate), poly (D,L-lactic acid) oligomers and polymers, poly(L-lacticacid) oligomers and polymers, poly(glycolic acid), copolymers of lacticacid and glycolic acid, poly(caprolactone), poly(valerolactone),polyanhydrides, copolymers of poly(caprolactone) or poly(lactic acid)For non-biologically related applications particularly preferredpolymeric blocks include polystyrene, polyacrylates, and butadienes.

After addition of these polymers or mixtures thereof, the resultingdispersed biologic particles in the polymer containing hydrophobicorganic solution phase can be formed into the desired microparticle ormonolith by techniques well known in the field. These includeemulsion-stripping techniques as described by Domb (U.S. Pat. No.5,578,325) or Gibson (U.S. Pat. No. 6,291,013 B1), spray drying, ormolding to form a solid matrix containing the encapsulated hydrophilicactive, such as a biologic. The matrix can be dried by spray drying, bypan drying, or by molding processes to obtain a solid final matrixcontaining the encapsulated active or biologic. Release is then effectedby dissolution, erosion, or swelling of the matrix phase. Thisencapsulation followed by matrix formation enables much higher loadingsof the hydrophilic active or biologic than can be achieved by simpledouble emulsion techniques which have been described previously in theliterature. These double emulsion routes suffer from burst release athigh loadings of the active in the matrix formulation.

Post Processing

Other post processing steps may also be carried out on the particles.Ligands may be conjugated to the surface of the particles. For uncoatedparticles, ligands are conjugated to the more non-polar blocks of thecopolymer. Depending on ligand size, conjugation may occur before orafter particle formation. For coated particles, ligands are conjugatedto the hydrophilic blocks of the coating amphiphilic copolymer.Depending on ligand size, conjugation may occur before or after particlecoating.

Particles may be freeze dried into a stable powder form. Particles maybe centrifuged out of solution and re-suspended in a new solvent inwhich they are still stable. Particles may be dialyzed to change thesolvent in which they are suspended or to remove small molecules.

Particles may be administered to a patient by any one of a variety ofmanners or a combination of varieties of manners. For example, particlesmay be administered orally, nasally, intraperitoneally, or parenterally,by intravenous, intramuscular, topical, or subcutaneous routes, or byinjection into tissue. The particles may be administered in apharmaceutically acceptable vehicle or carrier.

EXAMPLES Example 1: Particle Assembly and Morphology

To produce particles containing hydrophilic compounds, poly(n-butylacrylate)7.5 kDa-b-poly(acrylic acid)5.5 kDa (PBA-b-PAA) and thebiologic were first dissolved in a polar organic solvent such as DMSO ormethanol. In an FNP process, this stream was rapidly mixed with a lesspolar nonprocess solvent such as chloroform (CHCl₃) or acetone in a CIJmixer. The non-solvent caused the biologic to precipitate. Thisprecipitation was halted by the self-assembly of the hydrophilic PAAblock of the PAA-b-PBA on the growing particle surface. The finalparticles were sterically stabilized in the non-solvent by thehydrophobic PBA block. The resultant nanoparticle had a core-shellstructure.

This process has been applied to an assortment of model hydrophilicagents with varying molecular weights, including lysozyme as a modelprotein (14.3 kDa), the cyclic peptide antibiotic vancomycin (1.45 kDa),a model 7-amino acid long peptide(glycine-arginine-leucine-glycine-tryptophan-serine-phenylalanine(GRLGWSF), 822 Da), the dyes eosin Y (692 Da) and tartrazine (534 Da),the MRI contrast agent gadopentetic acid (548 Da), the aminoglycosideantibiotic tobramycin (468 Da), glutathione (307 Da), and tryptophan(204 Da). Each formulation resulted in nanoparticles with lowpolydispersities at a minimum loading of 50 wt %.

Example 2: Nanoparticle Loading and Size Control

The sizes of particles produced with FNP may be controlled through thetime scales for precipitation of the core material, as well as throughthe time scale for polymer self-assembly. These time scales aremodulated through material concentrations as well as through the choiceof nonprocess solvent.

Increasing the percentage of core material necessitated the formation oflarger particles. For both lysozyme and vancomycin, the particle sizeinitially decreased as biologic was added compared to the micelle sizeof the block copolymer. This may be because the addition of corematerial allows the PAA to more tightly pack due to charge shieldingeffects. Very high loadings of both lysozyme and vancomycin wereobtained. Loading of lysozyme as high as 75 wt % was obtained withoutthe formation of large precipitates, however, the particles were toolarge to analyze using dynamic light scattering (DLS). Vancomycinloadings up to 90 wt % resulted in stable particles, with sub-100 nmparticles observed for loadings below 80 wt %. These loadings indicatedthat, despite its high water solubility and poor solubility inchloroform, vancomycin played a role in stabilizing the surface—mostlikely through its aromatic groups.

Additional process variables that were found to impact the particle sizeincluded the total mass concentration of polymer and biologic in theDMSO stream, the volume fraction of acetone in the chloroform stream,and the volume fraction of water in the DMSO stream. The addition of 5vol % water in the DMSO stream reduced the size of the 50% loadedlysozyme particles from 125 nm to 45 nm. With the proper formulationparameters, nanoparticles with loadings greater than 50 wt % anddiameters less than 100 nm were readily accessible with the FNP process.By choosing a nonprocess solvent in which the biologic is negligiblysoluble the particles may have a very high (>90%) encapsulationefficiency.

Example 3: Stabilization of Nanoparticles for Further Processing

In order to stabilize the nanoparticles in aqueous environments andreduce the loss of encapsulated material during processing steps,methods of crosslinking the PAA shell in order to form a gel wereinvestigated. Ionic crosslinking was focused on because it reduces therisk of covalently modifying the encapsulated agent. The anionic PAAside groups may be crosslinked either with multivalent metal cations orpolyamines (S. Bontha, A. V. Kabanov, T. K. Bronich, Polymer micelleswith cross-linked ionic cores for delivery of anticancer drugs, J.Controlled Release. 114 (2006) 163-174; T. K. Bronich, A. V. Kabanov, V.A. Kabanov, K. Yu, A. Eisenberg, Soluble Complexes from Poly(ethyleneoxide)-block-polymethacrylate Anions and N-Alkylpyridinium Cations,Macromolecules. 30 (1997) 3519-3525; T. K. Bronich, P. A. Keifer, L. S.Shlyakhtenko, A. V. Kabanov, Polymer Micelle with Cross-Linked IonicCore, J. Am. Chem. Soc. 127 (2005) 8236-8237; R. T. Patil, T. J.Speaker, Retention of trypsin activity in spermine alginatemicrocapsules, J. Microencapsul. 14 (1997) 469-474). The nanoparticleswere successfully stabilized by including chloride salts of Ca²⁺, Zn²⁺,or Fe³⁺ in the nonprocess solvent stream. Spermine, which contains twoprimary and two secondary amines, and the positively charged antibiotictobramycin also stabilized the particles. Ionic gelation agents can beadded to the nanoparticle solution after the FNP process.

Iron III chloride, included at a 3:1 ratio of acid groups to iron, wasan especially effective crosslinking agent. Particles loaded with 50 wt% vancomycin and crosslinked with iron swelled minimally from 72 nm inCHCl₃ to 79 nm in methanol, which dissolves non-crosslinkednanoparticles. The minimum swelling indicated a small mesh size, whichis necessary to slow the release of encapsulated biologics. For example,the minimal swelling in methanol indicated tight crosslinking. The ironprovided enough electron contrast to allow for TEM imaging of thenanoparticles. A TEM micrograph of 20-40 nm particle cores supported theDLS data, which gave larger sizes because the measurement included thePBA shell.

Example 4: Coating Nanoparticles with PEG: Layer-by-Layer FNP

In order to produce nanoparticles that were sterically stabilized inaqueous media, the 50 wt % vancomycin particles stabilized with ironwere coated with PEG. The particles were suspended in acetone with onemass equivalent of polystyrene1.6 kDa-b-poly(ethylene glycol)5 kDa(PS-b-PEG). This stream was rapidly mixed with water in a second FNPstep. The PS block of the PS-b-PEG assembled on the collapsed PBAsurface of the particles. Without the PEG, the collapsed hydrophobic PBAsurface of the vancomycin nanoparticles caused them to aggregate inwater. The final PEG-stabilized particles were 85 nm with no visibleaggregates and a near neutral zeta potential. The final nanoparticlestructure was a tightly crosslinked hydrogel interior coated in ahydrophobic interface and sterically stabilized by a PEG brush.

The PEG-coated particles had an encapsulation efficiency of 40%(determined by high-pressure liquid chromatography) and a finalvancomycin loading of 11.5 wt %. Vancomycin is poorly soluble inchloroform and acetone, so the loss of material likely occurred in thecoating step. Because vancomycin has hydrophobic sections that played arole in stabilizing the particle surface in chloroform, it may not havebeen fully entrapped in the iron-PAA mesh.

Example 5: Nanoparticles Formed with Various Encapsulated Materials

Nanoparticles (NPs) with encapsulated hydrophilic model activepharmaceutical agents (APIs) were created using Flash NanoPrecipitation(FNP) using a confined impingement jets mixer (CIJ) which has beendescribed previously. Generally, the stabilizing polymer (PBA-b-PAA) andAPI were dissolved in a more polar organic solvent, typically consistingof MeOH or DMSO. This stream was rapidly mixed with an equal volumestream of a more nonpolar anti-solvent at equal flowrates, typicallyconsisting of CHCl₃ or acetone. The outlet stream of the CIJ wascollected in a stirring bath of antisolvent such that the finalnanoparticle solution is 90 vol % antisolvent. outlines the formulationsused in this study.

For crosslinked NPs, the crosslinking agent was included in theantisolvent stream such that the charge ratio (ratio of acid groups onthe PAA to positive charge of crosslinking agent) was 1:1.

Nanoparticles with a hydrophilic core (“inverse” nanoparticles) wereformed that encapsulated the small molecules tartrazine, eosin Y, andgadolinium-diethylene triamine pentaacetic acid (Gd-DTPA). The particlesize distribution for the sets of particles formed with theseencapsulated materials is shown in FIG. 3A.

Nanoparticles with a hydrophilic core were formed that encapsulated thebiologic antibiotics tobramycin and vancomycin. The particle sizedistribution for the sets of particles formed with these encapsulatedmaterials is shown in FIG. 3B.

Nanoparticles with a hydrophilic core were formed that encapsulated thesmall molecule biologics glutathione and tryptophan. The particle sizedistribution for the sets of particles formed with these encapsulatedmaterials is shown in FIG. 3C.

Nanoparticles with a hydrophilic core were formed that encapsulated thelarger biologics lysozyme and “Peptide I”(glycine-arginine-leucine-glycine-tryptophan-serine-phenylalanine(GRLGWSF)). The particle size distribution for the sets of particlesformed with these encapsulated materials is shown in FIG. 3D.

The experimental conditions used for the above-described encapsulationsystems are provided in Table 2, below.

TABLE 2 Nonprocess Sample Process Stream Stream Bath Lysozyme 500 uLDMSO, 500 uL CHCl₃ 4.5 mL 5 mg/mL lysozyme, CHCl₃ 5 mg/mL polymerVancomycin 500 uL DMSO with 500 uL CHCl₃ 4.5 mL 5 vol % MQ CHCl₃ 5 mg/mLlysozyme 5 mg/mL polymer pepI 500 uL DMSO with 500 uL CHCl₃ 4.5 mL 5 vol% MQ CHCl₃ 5 mg/mL pepI 5 mg/mL polymer Eosin Y* 500 uL DMSO, 500 uLCHCl₃ 4.5 mL 5 mg/mL Eosin Y, CHCl₃ 10 mg/mL polymer Tartrazine* 500 uLDMSO with 500 uL CHCl₃ 4.5 mL 5 vol % MQ, CHCl₃ 5 mg/mL tartrazine, 10mg/mL polymer Gd-DTPA** 500 uL DMSO with 500 uL CHCl₃ 4.5 mL 5 vol % MQ,CHCl₃ 5 mg/mL Gd-DTPA, 5 mg/mL polymer Tobramycin 500 uL DMSO, 500 uLDMSO, 4.5 mL 10 mg/mL polymer 5.5 mg/mL acetone tobramycin (1:1 chargeratio) Glutathione 500 uL DMSO with 500 uL CHCl₃ 500 uL 5 vol % MQ CHCl₃5 mg/mL glutathione 5 mg/mL polymer Tryptophan**** 500 uL DMSO with 500uL CHCl₃ 4.5 mL 5 vol % MQ and CHCl₃ 5 vol % acetic acid, 5 mg/mLtryptophan, 5 mg/mL polymer *PBA(3 kDa)-b-PAA(12 kDa) used in theseformulations, all others used PBA(7.5 kDa)-b-PAA(5.5 kDa) **API is firstdissolved in MQ (Milli-Q purified water) then diluted with DMSO ****APIis first dissolved in acetic acid, then diluted with DMSO

Among these encapsulated materials, the smallest encapsulated moleculehad a molecular weight of 186 Da, and the largest encapsulated molecularhad a molecular weight nearly two order of magnitude greater, 14 kDa,demonstrating the versatility of this process. The encapsulatedparticles also had a range of charges, for example, tartrazine isnegatively charged, whereas tobramycin is positively charged.

A person of skill in the art would appreciate that systems forencapsulating materials can be optimized to determine the best polarprocess solvent (e.g., DMSO vs. MeOH, optionally with additives),nonprocess solvent (antisolvent) (e.g., acetone, CHCl₃, toluene, orDCM), polymer/core material ratios, and/or water content of the solventstream.

Example 6: PAA-b-PBA Nanoparticles

Poly(acrylic acid)-b-poly(n-butyl acrylate) (12 kDa-b—3 kDa) wasdissolved in methanol at a concentration of 20 mg/mL. Basic chromiumacetate was dissolved in methanol at a mass concentration of 90 mg/mL.The solutions were mixed one part polymer solution to one part chromiumsolution by volume. Immediately after the mixture was prepared, it wasmixed 1:1 against a chloroform stream in a handheld confined impingementjet (CIJ) mixer. The effluent of the CIJ mixer was collected in arapidly stirring bath of chloroform such that the final solventcomposition was 1 part methanol to 9 parts chloroform by volume. Theparticles were mixed for 4 days to allow the chromium cations tocrosslink the poly(acrylic acid). The resulting particles were 135 nm inchloroform with a polydispersity index (PDI) less than 0.1. Swollen inmethanol, the resulting particles were 190 nm with a PDI less than 0.1.Particle diameter was measured by dynamic light scattering using aMalvern Zetasizer in normal analysis mode.

Example 7: Coating of PAA-b-PBA Nanoparticles with PS-b-PEG

The particles from Example 6 were diluted with acetone and centrifugedout of solution at 15000 rcf for 15 minutes. The supernatant wasdecanted and the pellet was resuspended in acetone. The particles werecentrifuged out of solution a second time at 15000 rcf for 15 minutes.The supernatant was decanted and the pellet was resuspended in acetoneto a final mass concentration of 6 mg/mL. Polystyrene-b-poly(ethyleneglycol) (1.6 kDa-b—5 kDa) was dissolved in THF at a mass concentrationof 12 mg/mL. The particle solution and block copolymer solution weremixed one to one by volume, and then it was mixed 1:1 against adeionized water stream in a handheld confined impingement jet (CIJ)mixer. In deionized water the resulting particles were 195 nm indiameter with a PDI of 0.1 as measured by dynamic light scattering usinga Malvern Zetasizer in normal analysis mode.

Example 8: PAA-b-PBA Nanoparticles with Glutathione

Poly(acrylic acid)-b-poly(n-butyl acrylate) (12 kDa-b—3 kDa) wasdissolved in methanol at a concentration of 40 mg/mL. Reducedglutathione (GSH) was dissolved in a 1:4 mixture (volume to volume)water and methanol at a mass concentration of 20 mg/mL. Basic chromiumacetate was dissolved in methanol at a mass concentration of 90 mg/mL.The solutions were mixed one part polymer solution to one part chromiumsolution to two parts glutathione solution by volume. Immediately afterthe mixture was prepared, it was mixed 1:1 against a chloroform streamin a handheld confined impingement jet (CIJ) mixer. The effluent of theCIJ mixer was collected in a rapidly stirring bath of chloroform suchthat the final solvent composition was 1 part water to 9 parts methanolto 90 parts chloroform by volume. The particles were mixed for 4 days toallow the chromium cations to crosslink the poly(acrylic acid). Theresulting particles were 110 nm in chloroform with a PDI less than 0.1.Swollen in methanol, the resulting particles were 130 nm with a PDI lessthan 0.1. Particle diameter was measured by dynamic light scatteringusing a Malvern Zetasizer in normal analysis mode.

Example 9: Coating of Glutathione-Containing PAA-b-PBA Nanoparticleswith PS-b-PEG

The particles from Example 8 were diluted with acetone and centrifugedout of solution at 15000 rcf for 15 minutes. The supernatant wasdecanted and the pellet was resuspended in acetone. The particles werecentrifuged out of solution a second time at 15000 rcf for 15 minutes.The supernatant was decanted and the pellet was resuspended in acetoneto a final mass concentration of 6 mg/mL. Polystyrene-b-poly(ethyleneglycol) (1.6 kDa-b—5 kDa) was dissolved in THF at a mass concentrationof 12 mg/mL. The particle solution and block copolymer solution weremixed one to one by volume, and then it was mixed 1:1 against adeionized water stream in a handheld confined impingement jet (CIJ)mixer. In deionized water the resulting particles were 160 nm indiameter with a PDI of 0.1 as measured by dynamic light scattering usinga Malvern Zetasizer in normal analysis mode.

Example 10: PAA-b-PEG Nanoparticles with Glutathione

Poly(acrylic acid)-b-poly(ethylene glycol) (5 kDa-b—5 kDa) was dissolvedin methanol at a concentration of 40 mg/mL. Reduced glutathione wasdissolved in a 1:4 mixture (volume to volume) water and methanol at amass concentration of 20 mg/mL. Basic chromium acetate was dissolved inmethanol at a mass concentration of 56 mg/mL. The solutions were mixedone part polymer solution to one part chromium solution to two partsglutathione solution by volume. Immediately after the mixture wasprepared, it was mixed 1:1 against a chloroform stream in a handheldconfined impingement jet (CIJ) mixer. The effluent of the CIJ mixer wascollected in a rapidly stirring bath of chloroform such that the finalsolvent composition was 1 part water to 9 parts methanol to 90 partschloroform by volume. The particles were mixed for 4 days to allow thechromium cations to crosslink the poly(acrylic acid). The particles werediluted with acetone and centrifuged out of solution at 15000 rcf for 15minutes. The supernatant was decanted and the pellet was resuspended inacetone. The particles were centrifuged out of solution a second time at15000 rcf for 15 minutes. The supernatant was decanted and the pelletwas resuspended in deionized water. The resulting particles were 245 nmin diameter with a PDI of 0.35 or lower, as measured by dynamic lightscattering using a Malvern Zetasizer in normal analysis mode.

Example 11

In a salinized vial, a solution of PS-b-PEG in acetone was added to coatPBA-b-PAA nanoparticles encapsulating vancomycin such that the massratio in solution was 2:1:1 PS-b-PEG to PBA-b-PAA to vancomycin. Thissolution was rotovapped to a total mass concentration of 5 mg/mL. Thenanoparticle solution was then diluted ten-fold with acetone and thenrotovapped back to a mass concentration of 5 mg/mL five times to ensureall chloroform had been removed.

The nanoparticle and PS-b-PEG solution were mixed at a 1:1 volume ratiowith Milli-Q purified water (MQ) in a CIJ and the exit stream wascollected in a stirring bath of MQ such that the final solution was 90vol % MQ. The process stream included 500 μL of 50 vol % DMSO, 45 vol %MeOH, and 5 vol % MQ, with 5 mg/mL of vancomycin and 5 mg/mL of polymer.The nonprocess stream included 600 μL of CHCl₃ and 50 μL of FeCl₃(crosslinking agent) in MeOH. The bath included 4.5 mL of CHCl₃.

The resulting particle sizes are shown in FIG. 4 for uncoated particlesin CHCl₃, uncoated particles in MeOH, and coated particles in water.

Hydrophobic polymer scaffolds, for example, those based on PLGA, canproduce particles capable of peptide and protein release over timescales greater than 1 month. Producing PLGA microparticles with highloading, high encapsulation efficiency, and controlled release canrequire the formation of a very fine and stable primary emulsioncontaining a high concentration of biologic. FNP can be used toencapsulate of biologics at loadings greater than 50 wt % innanoparticles less than 100 nm in diameter, much smaller than the poresproduced in most double emulsion methods. The FNP process is scalableand tunable—particles ranging from 40 nm to 300 nm were produced. Theresulting particles are sterically stabilized in polar organic solventsby a hydrophobic polymer shell. The anionic core has been crosslinkedwith multivalent cations and with polyamines. These particles may beincorporated into PLGA scaffolds in order to form a nanocompositematerial. The crosslinked hydrogel core can both increase overallencapsulation efficiency by reducing active pharmaceutical ingredient(API) loss to the outside aqueous phase during the final emulsificationstep, and provide an additional barrier to prolong the release of thetherapeutic and decrease the burst release. The dense hydrophobicpolymer brush can prevent coalescence and reduce channel formation,increasing the tortuosity of the final PLGA microparticles. A PAA blockcan protect the biologic from adsorption on the PLGA surface within thepore. The gels formed are nano-scale.

FNP can be used to formulate biologics in polymeric particles for drugdelivery. A delivery system can be tailored to optimize itsbiocompatibility. For example, stabilizing materials can include PLA (toreplace the PBA) and a biocompatible anionic polymer (to replace thePAA). Hyaluronic acid, poly(aspartic acid), and poly(glutamic acid) areall possible biodegradable alternatives to the PAA. The process can beoptimized to maintain the stability of biologics with more sensitivehigh levels of structure.

The references cited herein are incorporated by reference in theirentirety as if fully set forth herein.

The embodiments illustrated and discussed in this specification areintended only to teach those skilled in the art the best way known tothe inventors to make and use the invention. Nothing in thisspecification should be considered as limiting the scope of the presentinvention. All examples presented are representative and non-limiting.The above-described embodiments of the invention may be modified orvaried, without departing from the invention, as appreciated by thoseskilled in the art in light of the above teachings. It is therefore tobe understood that, within the scope of the claims and theirequivalents, the invention may be practiced otherwise than asspecifically described.

1. A method for encapsulating a water soluble agent comprising:dissolving the water soluble agent and a copolymer in a polar processsolvent to form a first process solution; and continuously mixing thefirst process solution with a nonprocess solvent to form a mixedsolution from which a nanoparticle assembles and precipitates, whereinthe copolymer comprises at least one region that is more polar and atleast one region that is less polar, wherein the nonprocess solvent isless polar than the polar process solvent, wherein the nanoparticlecomprises a core and a shell, wherein the core comprises the more polarregion of the copolymer and the water soluble agent, wherein the shellcomprises the less polar region of the copolymer, and wherein the mixingcauses no more than 20 percent by volume of the polar process solvent tophase separate from the mixed solution.
 2. The method of claim 1,wherein the water soluble agent is selected from the group consisting ofa biologic material, an amino acid, a peptide, a protein, DNA, RNA, asaccharide, glutathione, tryptophan, a lysozyme, glucagon-like peptide-1(GLP-1), a small molecule therapeutic, tobramycin, vancomycin, animaging agent, eosin, eosin Y, tartrazine, a metal chelate, a gadoliniumchelate, and gadolinium diethylene triamine pentaacetic acid (GD-DTPA).3. The method of claim 1, wherein the copolymer is selected from thegroup consisting of a random copolymer, a block copolymer, a diblockcopolymer, a triblock copolymer, a multiblock copolymer, and abranched-comb copolymer.
 4. The method of claim 1, wherein the at leastone more polar region of the copolymer comprises at least one anionicmore polar region.
 5. The method of claim 4, wherein the at least oneanionic more polar region comprises poly(acrylic acid) (PAA), hyaluronicacid, poly(glutamic acid), poly(aspartic acid), or combinations.
 6. Themethod of claim 1, wherein the at least one more polar region of thecopolymer comprises at least one cationic more polar region.
 7. Themethod of claim 6, wherein the at least one cationic more polar regioncomprises chitosan, histadine lipids, histamines, spermadines,polyethylene-imines, or combinations.
 8. The method of claim 1, whereinthe at least one less polar region of the copolymer comprisespoly(n-butyl acrylate) (PBA), poly(lactic acid) (PLA),poly(caprolactone) (PCL), poly(lactic-co-glycolic acid) (PLGA), orcombinations.
 9. The method of claim 1, wherein the copolymer ispoly(acrylic acid)-block-poly(n-butyl acrylate) (PAA-b-PBA).
 10. Themethod of claim 1, wherein the polar process solvent is selected fromthe group consisting of water, an alcohol, methanol, ethanol, acetone,acetonitrile, dimethyl sulfoxide (DMSO), dimethylformamide (DMF),N-methyl pyrrolidone (NMP), and combinations.
 11. The method of claim 1,wherein the polar process solvent is selected from the group consistingof methanol, DMSO, water, and combinations.
 12. The method of claim 1,wherein the nonprocess solvent is selected from the group consisting ofchloroform, dichloromethane, an alkane, hexane, an ether, diethyl ether,tetrahydrofuran (THF), toluene, acetone, and combinations.
 13. Themethod of claim 1, wherein the nonprocess solvent is selected from thegroup consisting of chloroform, acetone, and combinations.
 14. Themethod of claim 1, wherein the polar process solvent and the nonprocesssolvent are miscible.
 15. The method of claim 1, wherein a time ofmixing of the process solution with the nonprocess solvent is less thanan assembly time of the nanoparticle.
 16. The method of claim 1, whereinthe water soluble agent and the copolymer have a supersaturation levelin the solution ranging from 10 to 10,000.
 17. The method of claim 1,wherein the nanoparticle has a size ranging from about 40 nm to about400 nm.
 18. The method of claim 1, further comprising stabilizing thenanoparticle core through crosslinking of the copolymer.
 19. The methodof claim 18, wherein the nanoparticle is crosslinked during assembly ofthe nanoparticle.
 20. The method of claim 18, wherein the nanoparticleis crosslinked after assembly of the nanoparticle.
 21. The method ofclaim 18, wherein the crosslinking is covalent crosslinking.
 22. Themethod of claim 18, wherein the crosslinking is non-covalent, ionic,chelation, acid-base, or hydrogen bonding crosslinking.
 23. The methodof claim 18, wherein a crosslinking agent is added to crosslink aportion of the copolymer of anionic functionality and wherein thecrosslinking agent is selected from the group consisting of an alkalineearth halide, a magnesium halide, magnesium chloride, a calcium halide,calcium chloride, a transition metal halide, an iron halide, iron(III)chloride, spermine, and combinations.
 24. The method of claim 18,wherein a crosslinking agent is added to crosslink a portion of thecopolymer of anionic functionality and wherein the crosslinking agent isselected from the group consisting of a metal acetate, an alkaline earthacetate, a transition metal acetate, and calcium acetate.
 25. The methodof claim 18, wherein a crosslinking agent is added to crosslink aportion of the copolymer of anionic functionality and wherein thecrosslinking agent is chromium(III) acetate.
 26. The method of claim 18,wherein the water soluble agent comprises tobramycin and wherein thetobramycin crosslinks the copolymer.
 27. The method of claim 18, whereina crosslinking agent is added to crosslink a portion of the copolymer ofcationic functionality and wherein the crosslinking agent is selectedfrom the group consisting of polycitric acid, polyacrylic acid,polyaspartic acid, polyglutamic acid, multi-valent anions, andcombinations.
 28. The method of claim 1, further comprising coating thenanoparticle with an amphiphilic polymer, wherein the amphiphilicpolymer comprises at least one hydrophilic region and at least onehydrophobic region.
 29. The method of claim 28, comprising dissolvingthe amphiphilic polymer and suspending the nanoparticles in awater-miscible organic solvent to form a second process solution; andcontinuously mixing the second process solution with an aqueous solventto form a second mixed solution from which a coated nanoparticleassembles and precipitates, wherein the coated nanoparticle comprises acore, a shell, and a coating, wherein the coating comprises an innerregion and an outer region, wherein the inner region comprises the atleast one hydrophobic region of the amphiphilic polymer, wherein theouter region comprises the at least one hydrophilic region of theamphiphilic polymer.
 30. The method of claim 29, wherein the amphiphilicpolymer is selected from the group consisting of a random copolymer, agraft copolymer, a block copolymer, a diblock copolymer, a triblockcopolymer, and a multiblock copolymer.
 31. The method of claim 29,wherein the amphiphilic polymer is selected from the group consisting ofpolystyrene-block-poly(ethylene glycol) (PS-b-PEG), poly(lacticacid)-block-poly(ethylene glycol) (PLA-b-PEG),poly(caprolactone)-block-poly(ethylene glycol) (PCL-b-PEG),poly(lactic-co-glycolic acid)-block-poly(ethylene glycol) (PLGA-b-PEG),and poly(ethylene oxide)-block-poly(propylene oxide)-block-poly(ethyleneoxide) (PEO-b-PPO-b-PEO).
 32. The method of claim 29, wherein thewater-miscible organic solvent comprises tetrahydrofuran (THF) and/oracetone and wherein the aqueous solvent is water.
 33. A nanoparticlecomprising: a core comprising a more polar region of a copolymer and awater soluble agent and a shell comprising a less polar region of thecopolymer, wherein the more polar region of the copolymer in the core iscrosslinked.
 34. The nanoparticle of claim 33, further comprising acoating, wherein the coating comprises an inner region and an outerregion, wherein the inner region comprises a hydrophobic region of anamphiphilic polymer, wherein the outer region comprises a hydrophilicregion of the amphiphilic polymer.